Technical Abstract:
We developed two novel surveillance systems for “Candidatus Liberibacter” (CL) species detection and identification. The first system is called “single tube dual primer Taq-Man PCR” (STDP). The procedure involves two sequential rounds of PCR using the CL asiaticus species-specific outer and inner primer pairs. Different annealing temperatures between inner and outer pair primers allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The sensitivity of the dual primer system is comparable to conventional two-tube nested PCR, but the STDP eliminates the potential risk of cross contaminations commonly associated with conventional nested PCR. The second system is fluorescent dye-based Real-time PCR. This diagnostic system employs a pair of Liberibacter universal primers flanking a sequence region homologous to the known CL species; “CL asiaticus” (Las), “CL africanus” (Laf), “CL americanus” (Lam) and “CL solanacearum” (Lso). This primer system is able to quantitatively detect all four Liberibacter species but sequence variation among each species amplicon can be distinguished by performing a post-PCR high resolution melting curve analysis (HRMCA). Both systems reported here are robust and cost-effective for reliable detection, quantitation and identification of Liberibacter species in plants and insects and provide high throughput capabilities suitable for large scale year-round quarantine screening and epidemiological studies.